Molecular Ecology (2015) 24, 3467–3484 doi: 10.1111/mec.13248 Sky island diversification meets the multispecies coalescent – divergence in the spruce-fir moss spider (Microhexura montivaga, Araneae, Mygalomorphae) on the highest peaks of southern Appalachia MARSHAL HEDIN,* DAVE CARLSON* and FRED COYLE† *Department of Biology, San Diego State University, San Diego, CA 92182, USA, †PO Box 1935, Cullowhee, NC 28723, USA Abstract Microhexura montivaga is a miniature tarantula-like spider endemic to the highest peaks of the southern Appalachian mountains and is known only from six allopatric, highly disjunct montane populations. Because of severe declines in spruce-fir forest in the late 20th century, M. montivaga was formally listed as a US federally endangered species in 1995. Using DNA sequence data from one mitochondrial and seven nuclear genes, patterns of multigenic genetic divergence were assessed for six montane popula- tions. Independent mitochondrial and nuclear discovery analyses reveal obvious genetic fragmentation both within and among montane populations, with five to seven primary genetic lineages recovered. Multispecies coalescent validation analyses [guide tree and unguided Bayesian Phylogenetics and Phylogeography (BPP), Bayes factor delimitation (BFD)] using nuclear-only data congruently recover six or seven distinct lineages; BFD analyses using combined nuclear plus mitochondrial data favour seven or eight lineages. In stark contrast to this clear genetic fragmentation, a survey of sec- ondary sexual features for available males indicates morphological conservatism across montane populations. While it is certainly possible that morphologically cryptic speci- ation has occurred in this taxon, this system may alternatively represent a case where extreme population genetic structuring (but not speciation) leads to an oversplitting of lineage diversity by multispecies coalescent methods. Our results have clear conserva- tion implications for this federally endangered taxon and illustrate a methodological issue expected to become more common as genomic-scale data sets are gathered for taxa found in naturally fragmented habitats. Keywords: allopatry, Bayes factor delimitation, conservation genetics, montane speciation, population structure, unguided Bayesian Phylogenetics and Phylogeography Received 26 January 2015; revision received 13 May 2015; accepted 14 May 2015 mountains (Hedin 1997; Weisrock & Larson 2006; Keith Introduction & Hedin 2012). In each of these areas, mountains act as Evolutionary biologists have long been interested in naturally fragmented habitat islands, serve as refugia in evolution on mountains. In mainland North America, the face of climatic variation and/or generate strong conspicuous hotspots for montane diversification ecological gradients. These combinations of genetic and include the California Sierra Nevada (Rovito 2010; Scho- geographic isolation with potential selective differences ville & Roderick 2010; Hedin et al. 2013), ‘sky islands’ promote the evolution of both population genetic struc- of the desert southwest (Maddison & McMahon 2000; ture (arrays of geographically distinct populations Derkarabetian et al. 2011) and the southern Appalachian which are genetically divergent to various degrees) and clear species-level divergences. These naturally allopat- Correspondence: Marshal Hedin, Fax: +1 619 594 5676; ric systems also present classic difficulties for speciation E-mail: [email protected] © 2015 John Wiley & Sons Ltd 3468 M. HEDIN ET AL. biologists – when allopatry prevails and population has been suggested to potentially oversplit diversity in structure is ubiquitous, distinguishing population sub- dispersal-limited taxa (e.g. Niemiller et al. 2012; Barley division from speciation is challenging and often ‘fuzzy’ et al. 2013; McKay et al. 2013; Satler et al. 2013). Authors (Leavitt et al. 2007; Bond & Stockman 2008; Keith & of these methods have acknowledged the inherent diffi- Hedin 2012; Satler et al. 2013). For example, Hey (2009) culties associated with fragmented, allopatric systems pointed out that the null hypothesis for many species (e.g. Zhang et al. 2013). delimitation methods is a ‘no significant differentiation’ Microhexura montivaga, the spruce-fir moss spider, is a model. Because both population subdivision and specia- miniature mygalomorph spider (F. Dipluridae) endemic tion imply differentiation (i.e. rejection of the null), such to high-elevation red spruce-Fraser fir forests of the methods can mistakenly equate these potentially differ- southern Appalachian mountains (Coyle 1981, 1985). ent evolutionary dynamics (see also Hey & Pinho 2012). Recent survey work (Coyle 2009) indicates that Micro- Modern researchers have access to many types of hexura is distributed as six disjunct montane popula- data when investigating the interface between popula- tions, occupying the Virginia Balsam Mountains tion divergence and speciation, including data derived (Virginia), Roan Mountain (Tennessee/North Carolina), from morphology, behaviour, ecology and genomes. Grandfather Mountain (NC), the Black Mountains (NC), Assessing nuclear genomic divergence is particularly the Plott Balsam Mountains (NC) and the Great Smoky attractive in naturally fragmented systems because such Mountains (TN/NC) (Fig. 1). Most of these sky island data represent a common currency for measuring both populations are found above 1800 metres and include population genetic structure and speciation, and explicit the highest elevations in eastern North America (e.g. models are available that potentially distinguish popula- Mt. Mitchell, Black Mountains, NC). Spatial isolation tion structure from species-level divergence. These also potentially exists within mountain ranges, where include single-locus models (e.g. generalized mixed spiders build sheet webs underneath bryophyte mats Yule coalescent model, Pons et al. 2006; Fujisawa & Bar- on steep, north-facing rock outcrops (Coyle 2009). These raclough 2013) and arguably more powerful multilocus distinct outcrops are often separated by habitats lacking models. A plethora of multilocus species delimitation spiders (lower elevations, no spruce-fir forest, no rock methods have been developed over the past 10 years outcrops). Because of severe declines in spruce-fir forest (O’Meara 2010; Yang & Rannala 2010; Grummer et al. in the late 20th century, M. montivaga was formally 2014). Many empirical studies have been published listed as a US federally endangered species in 1995 (Fri- using such methods (reviewed in Fujita et al. 2012; Car- dell 1994, 2001). This status remains valid today, and stens et al. 2013), and this remains an active area of while recent survey work (Coyle 2009) has shown that method development (e.g. DISSECT, Jones et al. 2014; some montane populations include many rock outcrop unguided Bayesian Phylogenetics and Phylogeography demes and show comparatively large census population (BPP), Yang & Rannala 2014; *BFD, Leache et al. 2014). sizes (e.g. Great Smoky and Black Mountain popula- Despite this outstanding analytical progress, the tions), other populations are precariously small, known performance of multispecies coalescent methods in only from one or a few rock outcrops within a moun- highly genetically subdivided systems (e.g. taxa inhabit- tain range (e.g. Virginia Balsams, Plott Balsams). ing mountains, islands, caves) has not been extensively The research presented here was motivated primarily explored (Camargo & Sites 2013). A central assumption by an interest in documenting multilocus genetic struc- of many recently developed methods is the neutral coa- ture both within and among montane Microhexura pop- lescent (or something analogous), where gene trees ulations, with the goal of using this information to help evolve within species according to a no selection, no inform conservation decisions for this endangered recombination, panmixia model (Rannala & Yang 2003). species. There are no previous studies of Microhexura For example, the heuristic BROWNIE approaches devel- population structure, and while it is known that other oped by O’Meara (2010) simultaneously estimate spe- mygalomorphs (e.g. burrow-dwelling trapdoor spiders) cies trees and species limits by assuming unconstrained display remarkable microgeographic genetic differentia- gene flow within and a lack of gene flow between spe- tion (e.g. Bond & Stockman 2008; Hedin et al. 2013; cies. Simulations incorporating population structure Satler et al. 2013; Castalanelli et al. 2014; Opatova & tended to result in oversplitting by this method (O’Me- Arnedo 2014), whether such dispersal limitation applies ara 2010), and the empirical work of Niemiller et al. in the small-bodied (adult total length <6 mm), web- (2012) in naturally fragmented cavefishes hinted at building Microhexura is currently unknown. A second oversplitting, with both allelic and individual sampling goal was to explore the use of multilocus species delim- inflating species numbers. Another popular method is itation methods in this naturally fragmented system. BPP (Yang & Rannala 2010, 2014; Rannala & Yang 2013) The southern Appalachian mountains represent a hot- – this method assumes panmixia within species, and spot for speciation in both arthropods (e.g. Hedin 1997; © 2015 John Wiley & Sons Ltd SKY ISLAND SPECIES DELIMITATION 3469 Fig. 1 Geographic distribution of sam- pled specimens from six primary mon- tane populations. Number of sampled individuals and ‘demes’ (i.e. separate rock outcrop populations within moun-